Determination of gene dosage at the PMP22 and androgen receptor loci by quantitative PCR.

UNLABELLED: Although many genetic diseases are caused by the presence of point mutations in respective genes, an increasing number of diseases are known to be caused by gene copy number changes. We report the development of a rapid and reliable PCR-based method for quantitation of gene copy number with sufficient sensitivity to detect single copy changes without the use of radioactive or fluorescent labeling. The sensitivity of this technique has been demonstrated by the detection of the DNA duplication or deletion occurring in two inherited peripheral neuropathies, Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), that are caused by a reciprocal duplication or deletion event on chromosome 17p11.2-12. This method relies on the comparison of the amount of PCR product generated from a potentially duplicated or deleted target sequence with the amount of product generated from a disomic reference gene. The value of this ratio (target PCR product:reference PCR product) indicates whether the target sequence is duplicated, deleted, or unchanged. Using primers from within a duplicated or deleted region (PMP22 gene and EW401) and from within a reference region (NF1 gene), we tested 50 CMT1A, 30 HNPP, and 50 unaffected individuals for the presence of a DNA duplication or deletion. TARGET: reference ratios of 1.58, 1.02, and 0.56 were detected for the CMT1A, unaffected, and HNPP groups, respectively. Thus, differentiation of the three groups of individuals was on the basis of gene copy number. This technique was successfully used to detect the difference in the X chromosome copy number between males and females (target:reference ratios of 1.1 and 2.3, respectively). This approach to the detection of DNA duplications and deletions is sensitive, accurate, and has potential applications in the quantitation of changes in gene copy number associated with diseases characterized by such chromosomal alterations.

Apolipoprotein E genotyping in Alzheimer's disease in an Australian sample.

BACKGROUND: Several previous studies have reported an increased frequency of the E4 allele of the gene for Apolipoprotein (APOE4) in both familial and sporadic Alzheimer's disease (AD). We report the results of a study of this association in an Australian clinic-base sample. AIM: To investigate the relationship between APOE4 frequency and AD in an Australian clinic-based sample and compare the results with previous studies. METHODS: Subject DNA was PCR amplified, enzymatically digested with Hha1 and the resulting fragments electrophoretically separated. The genotypes were ascertained according to the resulting fragment sizes and the resulting allele frequencies analysed by calculating a z-statistic for comparison of two proportions. RESULTS: The frequency of the APOE4 allele was 53% in the AD group and 11% in the control group. This difference is statistically significant. There was no significant difference in E4 allele frequencies between AD subjects with a family history and those without. At least one E4 allele was found in 26/30 (87%) of AD patients and 10/50 (20%) of controls. The allele frequencies of the control subjects used in this study were found to be consistent with those of several previous studies. CONCLUSION: The frequency of the APOE4 allele was significantly higher in AD subjects than in unaffected controls. This provides further evidence of an association between APOE4 and both familial and sporadic AD.